A general approach for stabilizing nanobodies for intracellular expression

Conventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well documented examples of intracellular expression. Despite their promise as intracellular reagents, there has not been a systematic study of nanobody intracellular expression. Here, we examined intracellular expression of 75 nanobodies from the Protein Data Bank. Surprisingly, a majority of nanobodies were unstable in cells, illustrated by aggregation and clearance. Using comparative analysis and framework mutagenesis, we developed a general approach that stabilized a great majority of originally intracellularly unstable nanobodies, without compromising target-binding function. Additionally, we identified distinct sequence features impacting nanobody intracellular stability, and demonstrated mutationally stabilized nanobody expression in vivo, in the murine retina and in E. coli. This work will allow for standardized nanobody engineering for intracellular applications, potentiating a growing field of intracellular interrogation and intervention.